1. Field of the Invention
This invention is related to a method and assay kit for rapidly quantifying global DNA methylation through immobilizing DNA by simple dry-capture on the plastic carrier followed by immunodetection of 5-methylcytosine structure that is the marker of DNA methylation.
2. Description of the Related Art
DNA methylation is an epigenetic modification which is catalyzed by DNA cytosine-5-methyltransferases (DNMTs) and occurs at the 5-position (C5) of the cytosine ring, within CpG dinucleotides. DNA methylation is essential in regulating gene expression in nearly all biological processes including development, growth, and differentiation (Laird P W et al: Annu Rew. Genet, 1996; Reik W et al: Science, 2001; Robertson K D et al: Nature Rew. Genet, 2005). Alterations in DNA methylation have been demonstrated to cause the change in the gene expression. For example, hypermetlhylation leads to gene silencing or decreased gene expression while hypomethylation activates the genes or increases gene expression. Region-specific DNA methylation is mainly found in 5′-CpG-3′dinucleotides within the promoters or in the first exon of genes, which is an important pathway for the repression of gene transcription in diseased cells. Global DNA hypomethylation is likely caused by methyl-deficiency due to a variety of environmental influences, and has been proposed as a molecular marker in multiple biological processes such as cancer. It is well demonstrated that the decrease in global DNA methylation is one of the most important characteristics of cancer (Feinberg A P et al, Nature, 1983; Gama-Sosa M A et al, Nucleic Acids Res, 1983). Thus the determination of global methylation in cancer cells could provide very useful information for the detection and analysis of this disease. Many methods for the detection of global DNA methylation have been developed. These methods include: (1) high-performance liquid chromatography or thin-layer chromatography analysis (Breter H J et al: J chromatogr, 1976; Wagner I et. Al: Biochim Biophys Acta, 1981; Leonard S A et al: J chromatogr, 1993). In the analysis, DNA is digested into single nucleotides and total genomic 5-methylcytosine is quantified; (2) immunohistochemical staining in which tissue section is staining with anti-5-methylcytosine antibody to detect 5-methylcytosine positive cells (Hernandez-Blazquez F J et al: Gut, 2000; Piyaphilake C J et al: Dis Markers, 2005); (3) dot blot assay in which DNA is dot-blotted onto a nitrocellulose membrane followed by immunodetection of methylcytosine (Oakeley E. J et al: Proc. Natl. Acad. Sci. USA, 1997; Tao L et al: Toxic. Sci, 2005); (4) methyl accepting capacity or radiolabeled methyl incorporation (RMI) assay in which DNA is incubated with 3H—S-adenosylmethionine (3H—SAM) in the presence of methylase and RMI is inversely related to the degree of DNA methylation. (Wu J et al: Proc. Natl. Acad. Sci. USA, 1993; Belinsky S A et. al: Proc. Natl. Acad. Sci. USA, 1996); (5) analysis of repetitive DNA element methylation by PCR (Yang A S et al: Nucleic Acids Res, 2004). However these methods are labor intensive, time-consuming, or require large amounts of DNA (>250 ng) as the starting material for measurement, or rely on the use of expensive equipment and radioisotope reagents. These disadvantages lead to cost-ineffectiveness, low throughput and inconvenience for routine application in most health and medical institutions. Thus, there is a need for establishing a method to improve the detection of global DNA methylation.